Eukaryotic cells are subdivided by membranes into multiple functionally distinct compartments that are referred to as organelles. Each organelle includes proteins essential for its proper function. These proteins can include sequence motifs often referred to assorting signals. The sorting signals can aid in targeting the proteins to their appropriate cellular organelle(s). In addition, sorting signals can direct some proteins to be exported, or secreted, from the cell.
One type of sorting sequence is a signal sequence (also referred to as a signal peptide or leader sequence). The signal sequence is present as an amino-terminal extension on a newly synthesized polypeptide chain A signal sequence targets proteins to an intracellular organelle called the endoplasmic reticulum (ER).
The signal peptide takes part in an array of protein—protein and protein-lipid interactions that result in translocation of a polypeptide containing the signal sequence through a channel in the ER. After translocation, a membrane-bound enzyme (signal peptidase) liberates the mature protein from the signal sequence.
The ER functions to separate membrane-bound proteins and secreted proteins from proteins that remain in the cytoplasm. Once targeted to the ER, both secreted and membrane-bound proteins can be further distributed to another cellular organelle called the Golgi apparatus. The Golgi directs the proteins to vesicles, lysosomes, the plasma membrane, mitochondria and other cellular organelles.
Only a limited number of genes encoding human membrane-bound and secreted proteins have been identified. Examples of known secreted proteins include human insulin, interferon, interleukins, transforming growth factor-beta, human growth hormone, erythropoietin, lymphokines.
Transmembrane semaphoring SEMA6A is expressed in developing neural tissue. It is required for proper development of the thalamocortical projection. Critical to neuronal development and apoptosis is the transformation of extracellular signals to intracellular actions that result in cytoskeletal rearrangements. The protein family Ena/VASP plays an important role in actin and filament dynamics, while the semaphoring protein family act as guidance signals in embryogenesis and organogenesis.
Klostermann et al. identified novel transmembrane semaphoring, SEMA6A, in humans and mice (Klostermann et al., “The orthologous human and murine sepmaphorin 6A-1 proteins (SEMA6A-1/Sema6A-a) bind to the enabled/vasodilator-stimulated phosphoprotein-like protein (EVL) via a novel carboxyl-terminal zyxin-like domain,” 275 J. Biol. Chem. 39647-53 (2000)). The human SEMA6A gene is localized to chromosome 5q21-q22 and encodes a protein of 1,030 amino acids that has a calculated, approximate molecular mass of 112.2 kDa. The SEMA6A gene comprises 20 exons including 2 untranslated exons, and covers approximately 60 kb of the genomic sequence on chromosome 5. Human chromosome 5q21-q22 is known to be deleted in some forms of lung cancer.
Northern blot analysis reveals two 5- and 7-kb SEMA6A transcripts of equal intensity in human embryonic and adult tissues. Highest expression was observed in embryonic brain and kidney, whereas only low to moderate expression was seen in developing lung and liver. Small amounts of SEMA6A transcripts were detected in human adult tissues, with the exception of strong expression levels in highly regenerative placental tissues. Upon in situ hybridization, mouse neural embryonic tissues displayed high levels of Sema6a mRNA expression in proliferating zones in the diencephalon, retina, dorsal root ganglia, and trigeminal ganglion. SEMA6A directly links the Ena/VASP and semaphorin protein families because SEMA6A protein is capable of selective binding to the protein EVL (Ena/VASP-like protein). SEMA6A is colocalized with EVL via its zyxin-like C-terminal domain that contains a modified binding motif. Klostermann et al. concluded that their findings suggested a role for transmembrane semaphorins such as SEMA6A in retrograde signaling (Id.).
Leighton et al. identified an in vivo guidance function for semaphorin 6A (Leighton et al., “Defining brain wiring patterns and mechanisms through gene trapping in mice,” 410 Nature 174-9 (2001). SEMA6A belongs to a subfamily characterized by an extracellular semaphorin domain, a transmembrane domain, and a long cytoplasmic tail. Members of this class can repel sympathetic and dorsal root ganglion axons in vitro, consistent with a traditional role as a guidance signal. However, the length of the cytoplasmic tail, which includes an EVL-binding site in SEMA6A and an Src-binding site in SEMA6B, suggests that these semaphorins may also function as receptors.
Leighton et al. isolated an insertion in the SEMA6A gene at amino acid 473 in the semaphorin domain that completely abolishes wildtype Sema6A transcripts in a murine model (Id.). Homozygous mutant mice, both viable and fertile, displayed no obvious behavioral or morphologic phenotypes. Thalamocortical axons projected normally in heterozygotes. Strikingly however, in homozygotes, thalamocortical axons failed to turn up through the internal capsule and instead projected down toward the amygdala region. This defect was fully penetrant but specific to caudal thalamocortical axons; rostral projections appeared normal in every animal. That Sema6A is expressed in thalamocortical neurons and required for their axons to project properly suggests that the guidance defect is cell-autonomous and that Sema6A is acting in these axons as a guidance receptor. Sema6A is expressed broadly both in the thalamus and amygdala when these axons are growing, which made it impossible to distinguish fully between autonomous and nonautonomous activities without further experimentation. Leighton et al. proposed that, alternatively, Sema6A may function by promoting defasciculation of thalamocortical axons in a paracrine fashion, thereby enabling them to respond to other cues (Id.). Misrouting would then be secondary to a defasciculation failure.
The present invention discloses a novel protein encoded by a cDNA and/or by genomic DNA and proteins similar to it, namely, new proteins bearing sequence similarity to Semaphorin 6A precursor Splice variant, nucleic acids that encode these proteins or fragments thereof, and antibodies that bind immunospecifically to a protein of the invention. The novel protein(s) are an example of the continued search, identification and characterization of human secreted proteins and genes that encode them.